A prerequisite of life is the ability to maintain electrochemical imbalances across biomembranes and in all eukaryotes the plasma membrane potential and secondary transport systems are energised by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi1-3, and Na+,K+-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5. The plasma membrane proton pumps are included in the type III P-type ATPase subfamily while Na+,K+-ATPase and Ca2+-ATPase belong to the type II subgroup6. Electron microscopy has revealed the overall shape of proton pumps7, however an atomic structure has been lacking. Further data have been obtained by comparison on the primary structure with structural data obtained for a Ca2+ ATPase42, but this has not provided significant insight into the specific function of H+ proton pumps.
Proton translocating ATPases are essential for plant and fungi. Inhibitors of H+ ATPase therefore have applicability as herbicides and fungicides.
So far no selective H+-ATPase inhibitor has been identified. Vanadate is a potent inhibitor of plasma membrane H+-ATPases. Vanadate is a phosphate analogue that inhibits the pumps in its plus 5 valence state at low concentrations (μM range). Vanadate is however not selective for plasma membrane H+-ATPases as it inhibits all other P-type ATPases as well as many other enzymes which make use of ATP. For this reason there seem to be no therapeutic potential for vanadate.
Some specific inhibitors for other P-type ATPase have been identified, such as the cardiac glycosides which potently inhibit the Na+, K+ ATPase.
The H+/K+-ATPases mediates gastric acid secretion in animal cells when H+ is extruded in exchange for K+. Clinical blockage of H+/K+-ATPase pump activity is employed in the treatment of many human disease conditions such as dyspepsia, peptic ulcer disease, prevention of stress gastritis, gastrinomas and other conditions that cause hypersecretion of acid. Clinically used proton pump inhibitors are substituted pyridylmethylsulfinyl benzimidazole drugs. H+/K+-ATPase specific inhibitors Omeprazole, Lansoprazole, Esomeprazole and Pantoprazole are among the most selling drugs in the world. The inhibitors bind from the extracellular face of the enzyme to the transmembane domains of the protein. Hereby they restrain pump activity by blocking the ion transport pathway going trough transmembrane domains.
The Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase (SERCA) transport cytosolic calcium into intracellular compartments. Selective and potent inhibitors are known for SERCA (Inesi et al., 2005), and might have a therapeutic potential in prostate cancer (Denmeade and Isaacs, 2005). A plant derived sesquiterpene lactone, thapsigargin is highly effective in blocking SERCA with a Kd in the sub-nanomolar range. Thaspsigargin binds in a cavity bordered by transmembrane helix (M) M3, M5 and M7, part of the Ca2+ transport pathway, and blocks conformational transitions of the pumps. Other specific inhibitors are DBHQ (2,5-di(tert-butyl)hydroquinone), CPA (cyclopiazonic acid) and Br2-TITU (1,3-dibromo-2,4,6-tris (methyl-isothio-uronium)benzene). The DBHQ and CPA binding sites are close to the thapsigargin binding site, and the inhibitory mechanism is similar to that of thapsigargin. The binding site of TITU is not known.
Caloxins are specific inhibitors of plasma membrane Ca2+-ATPases (PMCA), and inhibition is established when caloxins binds to small extracellular domains of the pump molecule. Caloxins are peptide inhibitors, and are highly selective towards PMCA's. PMCA's extrude calcium from the cells, and defects in the activity of these pumps have been demonstrated to be associated with hypertension and decreased sperm mobility. Caloxins could potentially be used as contraceptive agents or to modulate artery blood pressure.
The SERCA-type Ca2+-ATPase of the malaria-inducing parasite Plasmodium falciparum has been pinpointed as the target of the widely used anti-malarial drug artemisinin. Mutational studies suggest that the binding site is near that of thapsigargin and that the inhibitory function is similar (review: Golenser J, Waknine J H, Krugliak M, Hunt N H, Grau G E., Current perspectives on the mechanism of action of artemisinins. Int J Parasitol. 2006 December; 36(14):1427-41)
As seen from the above specific inhibitors of different ATPases have various applications. Until now no specific inhibitors of the H+-ATPases are known. This may be accounted for by the lack of knowledge regarding the functionality of the H+ATPase. Several studies have aimed ad clarifying the overall structure of H+ ATPase based on structural data obtained from different ATPases, such as the SERCA (Ca2+ pump) as mentioned above. The structural models have been useful for identifying areas of similarity but the areas which are different, and therefore expected to be responsible for the selectivity of the pumps can not be envisioned with a sufficient level of detail from these model structures. Thus so far no selective H+ pump inhibitors have been identified.
In order to solve this problem the availability of high quality structural data of a H+pump may be of great help.
The previous lack of structural data of sufficient quality can be attributed to the difficulties encountered when expressing, purifying and crystallizing trans-membrane proteins and in particular complex proteins as type III P-type ATPase's.
Inhibitors of fungi H+ ATPases may be used in the treatment of fungal infections.
Inhibitors of H+ ATPases further have applications in agricultural industry as weed killers (herbicides) and fungicides.